The Tudor-domain protein TDRD7, mutated in congenital cataract, controls the heat shock protein HSPB1 (HSP27) and lens fiber cell morphology

Mutations of the RNA-granule component TDRD7 (OMIM: 611258) cause pediatric cataract in humans. Here, we applied an integrated approach to elucidate the molecular pathology of cataract in Tdrd7 targeted-knockout (Tdrd7-/-) mice. Tdrd7-/- animals precipitously develop lens fiber cell abnormalities early in life, suggesting a global-level breakdown/mis-regulation of key cellular processes. High-throughput RNA-sequencing followed by iSyTE-integrated bioinformatics-based analysis identified the molecular chaperone and cytoskeletal-modulator, HSPB1 (HSP27), among the high-priority down-regulated candidates in Tdrd7-/- lens. Moreover, a protein 2-D fluorescence difference gel electrophoresis-coupled mass spectrometry screen also identified HSPB1 to be reduced in Tdrd7-/- lens, offering independent support for focusing efforts on this factor to explain Tdrd7-/- cataract. Reduction of HSPB1 preceded lens morphological abnormalities, suggesting that cytoskeletal defects underlie the Tdrd7-/- cataract phenotype. In agreement, scanning electron microscopy revealed abnormal fiber cell membrane protrusions in Tdrd7-/- lenses. Significantly, abnormal F-actin staining was detected specifically in Tdrd7-/- fiber cells that exhibit nuclear degradation, thereby revealing that there are distinct mechanisms based on pre- or post-nuclear degradation differentiation stage for F-actin cytoskeletal maintenance in fiber cells. Further, RNA-immunoprecipitation identified Hspb1 mRNA in wild-type lens lysate TDRD7-pulldowns, and single-molecule RNA-imaging showed co-localization of TDRD7 protein with cytoplasmic Hspb1 mRNA in a specific pre-nuclear degradation area of differentiating fiber cells, indicating that TDRD7-ribonucleoprotein complexes are necessary for controlling optimal levels of key factors in lens development. Together, these data uncover a novel role for TDRD7 in regulating elevation of stress-responsive chaperones for cytoskeletal maintenance in post-nuclear degradation lens fiber cells, perturbation of which causes early-onset cataracts. For RNA-sequencing (RNA-Seq) experiments, mouse lenses at stage P4 (n = 15 lenses per biological replicate) were collected from Tdrd7-/- or control mice. RNA isolation was performed using the mirVanaTM RNA isolation kit (Life Technologies, Grand Island, NY). Total RNA isolation, followed by removal of small molecular weight RNA was performed according to manufacturer's instructions. Library was prepared using TruSeq RNA Library Prep Kit v2 (Illumina), and sequencing was performed on a 2x75 paired end run using standard protocols on an Illumina HiSeq 2500 sequencing system. Briefly, mRNA was purified from the total RNA samples using Oligo dT conjugated magnetic beads, converted to adaptor-tagged, paired-end fragments which were then used for cluster generation onto a TruSeq v3 flow cell according to the Illumina® TruSeq RNA Sample Preparation Kit v2. Sequencing was carried out using the SBS Sequencing Kit. Images were analyzed using the Illumina Pipeline software (version RTA 1.13.48/CASAVA 1.8.2), and bases were called and translated to generate FASTQ sequence files.RNA颗粒组分TDRD7（OMIM: 611258）的突变可引发人类儿童期白内障。本研究采用整合解析策略，阐明了Tdrd7靶向敲除（Tdrd7-/-）小鼠白内障的分子病理机制。Tdrd7-/-小鼠在生命早期即骤然出现晶状体纤维细胞异常，提示关键细胞过程发生了全局性紊乱或调控失常。 高通量RNA测序（RNA-sequencing, RNA-Seq）结合基于iSyTE的整合生物信息学分析，筛选出分子伴侣兼细胞骨架调节因子HSPB1（HSP27），为Tdrd7-/-晶状体中高优先级下调的候选靶点之一。此外，二维荧光差异凝胶电泳偶联质谱筛选实验同样证实，HSPB1在Tdrd7-/-晶状体中表达降低，为聚焦该因子阐释Tdrd7-/-白内障的发病机制提供了独立佐证。 HSPB1的表达下调早于晶状体形态学异常，提示细胞骨架缺陷是Tdrd7-/-白内障表型的核心致病基础。扫描电子显微镜观察进一步证实，Tdrd7-/-晶状体中存在异常的纤维细胞膜突起。值得注意的是，异常F-肌动蛋白（F-actin）染色仅在发生核降解的Tdrd7-/-纤维细胞中被检测到，这表明在晶状体纤维细胞的不同分化阶段（核降解前或核降解后），F-肌动蛋白细胞骨架的维持存在基于分化时序的独特调控机制。 进一步的RNA免疫沉淀（RNA-immunoprecipitation, RIP）实验显示，野生型晶状体裂解液经TDRD7下拉富集的产物中存在Hspb1 mRNA；单分子RNA成像结果表明，TDRD7蛋白与细胞质Hspb1 mRNA在分化纤维细胞的特定核降解前区域发生共定位，提示TDRD7-核糖核蛋白复合物对于维持晶状体发育过程中关键因子的最优表达水平不可或缺。 综上，本研究揭示了TDRD7在核降解后晶状体纤维细胞中，通过调控应激响应分子伴侣的表达以维持细胞骨架稳态的全新功能；该功能的扰动可导致早发性白内障。 针对RNA测序（RNA-sequencing, RNA-Seq）实验，我们从Tdrd7-/-或对照小鼠中收集P4期晶状体（每个生物学重复包含15个晶状体）。采用mirVana™ RNA分离试剂盒（Life Technologies，美国纽约州格兰德岛）完成总RNA提取，并依照制造商说明书去除小分子RNA。使用TruSeq RNA Library Prep Kit v2（Illumina）构建测序文库，随后按照标准流程在Illumina HiSeq 2500测序系统上开展2×75 bp双端测序。 简要实验流程如下：利用寡聚dT偶联磁珠从总RNA样品中纯化mRNA，将其转化为带有接头标签的双端测序片段，随后依照Illumina® TruSeq RNA Sample Preparation Kit v2的操作指南，在TruSeq v3流动槽上完成簇生成。使用SBS测序试剂盒进行上机测序，通过Illumina Pipeline软件（版本RTA 1.13.48/CASAVA 1.8.2）分析测序图像，完成碱基识别与序列转换，最终生成FASTQ格式序列文件。